Alteration of long- and short-term hematopoietic stem cell ratio causes myeloid-biased hematopoiesis
- Hematopoietic Stem Cell Biology and Medical Innovation (HSCBMI), Department of Pediatrics, Kobe University Graduate School of Medicine, Japan
- RIKEN Center for Biosystems Dynamics Research, Japan
- Weill Cornell, Rockefeller, Sloan-Kettering, Tri-Institutional MD-PhD Program, United States
- Institute of Laboratory Animals, Kyoto University Graduate School of Medicine, Japan
- Department of Pediatrics, Kobe University Graduate School of Medicine, Japan
- Department of Hematology and Oncology, Kyoto University Graduate School of Medicine, Japan
Figures
Figure 1 with 1 supplement
Comprehensive analysis of hematopoietic alternations with age shows a discrepancy of age-associated changes between peripheral blood and bone marrow (BM).
(A) Average frequency of myeloid cells (neutrophils and monocytes) and lymphoid cells (B cells, T cells, and NK cells) in PB at the age of 2 and 3 months (n = 6), 6 months (n = 6), 12 and 13 months (n = 6), 18 months (n = 6), 20 and 21 months (n = 5), and ≥23 months (n = 16). Abbreviation: PB = peripheral blood. (B) Average frequency of immunophenotypically defined hematopoietic stem cell (HSC) and progenitor cells in BM of 2- to 3-month mice (n = 6), 6-month mice (n = 6), 12- to 13-month mice (n = 6), and ≥23-month mice (n = 7). (C) Age-associated changes of immunophenotypically defined HSC and myeloid differentiation components (CMP and myeloid cells in the PB). The ratio of aged to young frequency was calculated as (the fraction frequency at each aged mice (%))/(the average fraction frequency at 2- to 3-month mice (%)). *p < 0.05, **p < 0.01, ***p < 0.001. Data and error bars represent means ± standard deviation.
Figure 1—figure supplement 1
Gating scheme for hematopoietic cells.
(A) Gating scheme to identify peripheral blood (PB) cell types (NK cell, neutrophil, monocyte, T cell, and B cell) after exclusion of doublets and dead cells. (B) Representative flow cytometry gating to isolate immunophenotypically defined hematopoietic stem cell (HSC), multipotent progenitor (MPP), Flk2+ progenitor (Flk2+), LKS after exclusion of doublets and dead cells, and lineage marker positive cells. (C) Representative flow cytometry gating to isolate common lymphoid progenitor (CLP), granulocyte–macrophage progenitor (GMP), common myeloid progenitor (CMP), and megakaryocyte–erythrocyte progenitor (MEP) after exclusion of doublets and dead cells, and lineage marker positive cells.
Figure 2 with 1 supplement
The expansion of myeloid-biased clones was not observed in 2-year-old long-term hematopoietic stem cells (LT-HSCs) after their transplantation.
(A) Hoxb5 reporter expression in bulk-hematopoietic stem cell (HSC), MPP, Flk2+, and Lin−Sca1−c-Kit+ populations in the 2-year-old Hoxb5-tri-mCherry mice (Upper panel) and 3-month-old Hoxb5-tri-mCherry mice (lower panel). Values indicate the percentage of mCherry+ cells ± standard deviation in each fraction (n = 3). (B) Experimental design to assess the long-term reconstitution ability of Hoxb5+ or Hoxb5− HSCs. Hoxb5+ and Hoxb5− HSCs were isolated from 2-year-old CD45.2 Hoxb5-tri-mCherry mice and were transplanted into lethally irradiated CD45.1 recipient mice with 2 × 105 supporting cells (Hoxb5+ HSCs, n = 13; Hoxb5− HSCs, n = 10). For secondary transplants, 1 × 107 whole bone marrow (BM) cells were transferred from primary recipient mice. Abbreviations: PB = peripheral blood, RT = radiation therapy. (C) Percentage chimerism at 16 weeks after receiving 10 aged Hoxb5− HSCs or 10 aged Hoxb5+ HSCs. Each column represents an individual mouse. (D) Percentage chimerism at 16 weeks after whole BM secondary transplantation. Donor whole BM cells for secondary transplantation were taken from mice denoted by † in (C). (E) Kinetics of average donor chimerism in each PB fraction after primary transplantation. (F) Kinetics of average donor chimerism after secondary transplantation. (G) Kinetics of average donor myeloid output myeloid proportion in donor cells in LT-HSC recipient mice after primary and secondary transplantation. *p < 0.05, **p < 0.01, ***p < 0.001. Data and error bars represent means ± standard deviation.
Figure 2—figure supplement 1
Aged Hoxb5+ hematopoietic stem cells (HSCs) retain robust hematopoietic capacity.
(A) Percentage chimerism at 4, 8, and 12 weeks after receiving 10 aged Hoxb5− HSCs or 10 aged Hoxb5+ HSCs after primary transplantation. (B) Percentage chimerism at 4, 8, and 12 weeks after receiving 10 aged Hoxb5− HSCs or 10 aged Hoxb5+ HSCs after secondary transplantation.
Figure 3 with 1 supplement
Aged long-term hematopoietic stem cells (LT-HSCs) show balanced hematopoiesis throughout life.
(A) Experimental design for competitive co-transplantation assay using young LT-HSCs sorted from Hoxb5-tri-mCherry GFP mice and aged LT-HSCs sorted from Hoxb5-tri-mCherry mice. Ten CD45.2+ young LT-HSCs and 10 CD45.2+ aged LT-HSCs were transplanted with 2 × 105 CD45.1+/CD45.2+ supporting cells into lethally irradiated CD45.1+ recipient mice (n = 8). (B) Lineage output of young or aged LT-HSCs at 4, 8, 12, and 16 weeks after transplantation. Each bar represents an individual mouse. (C) Lineage output kinetics of young LT-HSCs or aged LT-HSCs at 4, 8, 12, and 16 weeks post-transplant. (D) Competitive analysis of young LT-HSCs versus aged LT-HSCs lineage output at 4, 8, 12, and 16 weeks post-transplant. The competitive ratio was calculated as the proportion of young LT-HSC-derived cells versus aged LT-HSC-derived cells in each fraction. Abbreviations: Neut = neutrophils, Mo = monocytes, B = B cells, T = T cells, and NK = NK cells. Data and error bars represent means ± standard deviation. ‘n.d.’ stands for ‘not detected’.
Figure 3—figure supplement 1
Aged bulk-hematopoietic stem cells (HSCs) show myeloid-biased hematopoiesis compared to young bulk-HSCs.
(A) Experimental design for competitive co-transplantation assay using young bulk-HSCs sorted from 3-month-old Hoxb5-tri-mCherry GFP mice and aged bulk-HSCs sorted from 24-month-old Hoxb5-tri-mCherry mice. Ten CD45.2+ young bulk-HSCs and 10 CD45.2+ aged bulk-HSCs were transplanted with 2 × 105 CD45.1+/CD45.2+ supporting cells into lethally irradiated CD45.1+ recipient mice (n = 10). (B) Lineage output of young or aged bulk-HSCs at 4, 8, 12, and 16 weeks after transplantation. Each bar represents an individual mouse. (C) Lineage output kinetics of young bulk-HSCs or aged bulk-HSCs at 4, 8, 12, and 16 weeks post-transplant. (D) Competitive analysis of young bulk-HSCs versus aged bulk-HSCs lineage output at 4, 8, 12, and 16 weeks post-transplant. The competitive ratio was calculated as the proportion of young bulk-HSC-derived cells versus aged bulk-HSC-derived cells in each fraction. Abbreviations: Neut = neutrophils, Mo = monocytes, B = B cells, T = T cells, and NK = NK cells. *p < 0.05, **p < 0.01. Data and error bars represent means ± standard deviation. ‘n.d.’ stands for ‘not detected’.
Figure 4 with 1 supplement
Myeloid-associated genes were not enriched in aged long-term hematopoietic stem cells (LT-HSCs) compared to their young counterparts.
(A) Experimental schematic for transcriptome analysis. LT-HSCs (n = 3), short-term hematopoietic stem cells (ST-HSCs) (n = 3), and bulk-hematopoietic stem cells (HSCs) (n = 3) were sorted from young (2–3 months) or aged (23–25 months) Hoxb5-tri-mCherry mice, after which each RNA was harvested for RNA sequencing. (B) Hierarchical clustering dendrogram of whole transcriptomes using Spearman distance and the Ward clustering algorithm. (C) Violin plots showing normalized gene expression levels for each gene set in young and aged LT-HSCs, ST-HSCs, and bulk-HSCs. Expression values for each gene were standardized independently by applying Z score transformation. (D, E) Venn diagram showing the overlap of genes between three myeloid signature gene sets and lymphoid signature gene sets (Sanjuan-Pla et al., 2013; Pronk et al., 2007; Chambers et al., 2007). (F, G) Signature enrichment plots from gene set enrichment analysis (GSEA) using defined myeloid and lymphoid signature gene sets that overlapped in the three gene sets. Values indicated on individual plots are the normalized enrichment score (NES) and q-value of enrichment. ***p < 0.001.
Figure 4—figure supplement 1
Compared to their respective young controls, aged bulk-hematopoietic stem cells (HSCs) exhibit greater enrichment of myeloid gene expression than aged long-term hematopoietic stem cells (LT-HSCs).
(A) Average expression level of Hoxb5 in young and aged LT-, ST-, and bulk-HSCs (n = 3). (B) The ratio of myeloid gene expression of aged cells to young cells. The ratio of each gene expression was calculated as (the average read count in aged cells)/(the average read count in young cells). (C) Signature enrichment plots from gene set enrichment analysis (GSEA) using previously reported pre-granulocyte/macrophage progenitor and myeloid signature gene sets. The values indicated on individual plots are the normalized enrichment score (NES) and q-value of the enrichment. (D) Signature enrichment plots from GSEA analysis using previously reported common lymphoid progenitor (CLP) and lymphoid signature gene sets. The values indicated on individual plots are the normalized enrichment score (NES) and q-value of the enrichment.
Figure 5 with 1 supplement
The memory-type lymphocytes in the peripheral blood (PB) make it look as if short-term hematopoietic stem cells (ST-HSCs) are lymphoid-biased hematopoietic stem cells (HSCs).
(A) Experimental design for assessing the lineage output of young long-term hematopoietic stem cells (LT-HSCs) or ST-HSCs. Ten LT-HSCs or 10 ST-HSCs were isolated from 2-month-old CD45.2 Hoxb5-tri-mCherry GFP mice and were transplanted into lethally irradiated CD45.1 recipient mice with 2 × 105 supporting cells (LT-HSCs, n = 3; ST-HSCs, n = 4). (B) Kinetics of average frequency of lymphoid cells (B cells, T cells, and NK cells) in donor fraction after LT-HSC or ST-HSC transplantation. (C) Gating scheme to identify memory (central and effector) T cells and naive T cells in the PB after excluding doublets, dead cells, and non-donor cells. (D) Percentage of memory (central and effector) T cells and naive T cells in donor CD4+ fraction 10 months after LT-HSC or ST-HSC transplantation. (E) Gating scheme to identify donor cells in bulk-HSC fraction in bone marrow analysis. (F) Donor chimerism in bulk-HSC fraction 12 months after LT-HSC or ST-HSC transplantation. *p < 0.05, ***p < 0.001. Data and error bars represent means ± standard deviation.
Figure 5—figure supplement 1
Short- and long-term hematopoietic stem cell (ST-HSC and LT-HSC) ratio within bulk-hematopoietic stem cell (HSC) fraction changes with age.
Age-associated alternation of the average frequency of LT-HSCs and ST-HSCs in the bulk-HSC fraction at the age of 2- to 3-month mice (n = 6), 6-month mice (n = 6), 12-month mice (n = 6), and ≥23-month mice (n = 16). Error bars represent standard deviation. ***p < 0.001.
Figure 6 with 1 supplement
Hematopoiesis after transplantation inclined either toward myeloid or lymphoid cell production by artificially changing the ratio of long-term hematopoietic stem cell (LT-HSC)/short-term hematopoietic stem cell (ST-HSC).
(A) Experimental design for the transplantation of 2- to 3-month-old LT-HSCs and ST-HSCs in a 2:8 ratio (the same ratio as in young mice bone marrow [BM]) or 5:5 ratio (the same ratio as in aged mice BM). Donor cells were transplanted with 2 × 105 CD45.1+/CD45.2+ supporting cells into lethally irradiated CD45.1+ recipient mice (2:8 ratio, n = 18; 5:5 ratio, n = 23). (B) Donor lineage output of young LT-HSC and ST-HSC transplanted either in a 2:8 ratio or a 5:5 ratio at 4, 8, 12, and 16 weeks post-transplant. Each bar represents an individual mouse. (C) Kinetics of average lineage output of young LT-HSCs and ST-HSCs in a 2:8 ratio or a 5:5 ratio at 4, 8, 12, and 16 weeks post-transplant. (D) Frequency of myeloid cells in donor cell fraction. *p < 0.05, **p < 0.01. Error bars represent standard deviation. Data represent two independent experiments.
Figure 6—figure supplement 1
Changing the short-term hematopoietic stem cell (ST-HSC)/long-term hematopoietic stem cell (LT-HSC) ratio accelerates lymphoid hematopoiesis in the use of aged donor cells.
(A) Experimental design for the transplantation of aged LT-HSCs and ST-HSCs in a 5:5 ratio (the same ratio as in aged mice bone marrow [BM]) or a 2:8 ratio (the same ratio as in young mice BM). Donor cells were transplanted with 2 × 105 CD45.1+/CD45.2+ supporting cells into lethally irradiated CD45.1+ recipient mice (5:5 ratio, n = 16, 2:8 ratio, n = 15). (B) Donor lineage output of aged LT-HSCs and ST-HSCs transplanted either in a 5:5 ratio or a 2:8 ratio, 4, 8, 12, and 16 weeks post-transplantation. Each bar represents an individual mouse. (C) Kinetics of average lineage output of aged LT-HSCs and ST-HSCs in a 5:5 ratio or a 2:8 ratio at 4, 8, 12, and 16 weeks post-transplantation. (D) Frequency of lymphoid cells in donor cell fraction. *p < 0.05. Error bars represent standard deviation.
Figure 7
Age-associated physiological changes drive differentiation of long-term hematopoietic stem cells (LT-HSCs) toward myeloid cells.
(A) Experimental design for assessing the impact of age-associated physiological changes on differentiation of LT-HSCs. Ten GFP+ LT-HSCs sorted from young (2–3 months) Hoxb5-tri-mCherry GFP mice were transplanted with 2 × 105 CD45.1+/CD45.2+ supporting cells into lethally irradiated young or aged recipient mice. We defined donor cells as GFP+ cells and supporting cells as CD45.1+/CD45.2+ cells. (B) Survival rate of recipient mice in each group. (C) Donor lineage output in young or aged recipient mice 11–12 weeks after transplanting young LT-HSCs (young recipient, n = 17; aged recipient, n = 10). (D) Myeloid output (frequency of donor myeloid cells in donor fraction) in young or aged recipient mice 11–12 weeks after transplantation. (E) Kinetics of lineage output from donor LT-HSCs in young or aged recipient mice 4, 8, 11, and 12 weeks after transplantation. (F) Average frequency of donor bulk-hematopoietic stem cell (HSC) and progenitor cells in donor bone marrow (BM) live cells (young recipient, n = 8; aged recipient, n = 8). BM samples were taken from mice denoted by † in (C). (G) Representative immunofluorescence images of frozen spleen sections derived from young or aged recipient mice. Green: donor cells (GFP fluorescence); blue: DNA (4′,6-diamidino-2-phenylindole, DAPI); scale bar: 200 μm. (H) Frequency of donor cells in spleen B cells of young or aged recipient mice (young recipient, n = 8; aged recipient, n = 8). Spleens are taken from mice denoted by † in (C). (I) Representative immunofluorescence images of frozen thymus sections derived from young or aged recipient mice. Green: donor cells (GFP fluorescence); blue: DNA (DAPI); scale bar: 200 μm. (J) Frequency of donor cells in thymus T cells of young or aged recipient mice (young recipient, n = 8; aged recipient, n = 8). Thymi are taken from mice denoted by † in (C). *p < 0.05, ***p < 0.001. Error bars represent standard deviation. Data represent two independent experiments.
Figure 8 with 1 supplement
Our new model: self-renewal heterogeneity model.
It has been thought that there were myeloid (My-) or lymphoid biased (Ly-) hematopoietic stem cells (HSCs), and that clonal selection of My-HSCs caused age-associated myeloid-biased hematopoiesis. However, in our model, long-term hematopoietic stem cells (LT-HSCs) represent unbiased hematopoiesis throughout life. Short-term hematopoietic stem cells (ST-HSCs) lose their hematopoietic ability within a short period, and memory-type lymphocytes remain in the peripheral blood (PB) after ST-HSC transplantation. These remaining memory-type lymphocytes make it look as if ST-HSCs are lymphoid-biased (the upper section). As a result, the age-associated relative decrease of ST-HSCs in bulk-HSC fraction causes myeloid-biased hematopoiesis with age. Additionally, the blockage of lymphoid differentiation at the spleen and thymus accelerates further myeloid-biased hematopoiesis in aged mice (the lower section).
Figure 8—figure supplement 1
Analysis of donor chimerism in whole blood cells for each transplantation assay.
(A) Donor chimerism analysis related to Figure 2C, E. (B) Donor chimerism analysis related to Figure 2D, F. (C) Donor chimerism analysis related to Figure 3B, C. (D) Donor chimerism analysis related to Figure 3—figure supplement 1B-C. (E) Donor chimerism analysis related to Figure 5B. (F) Donor chimerism analysis related to Figure 6B, C. (G) Donor chimerism analysis related to Figure 7C–E. Donor chimerism was calculated as the frequency of donor cells in whole peripheral blood. *p < 0.05, **p < 0.01, ***p < 0.001. Data and error bars represent means ± standard deviation.
Author response image 1
(A) Experimental design for competitive co-transplantation assay. Ten CD45.2+ young LT-HSCs and ten CD45.2+ aged LT-HSCs were transplanted with 2 × 105 CD45.1+/CD45.2+ supporting cells into lethally irradiated CD45.1+ recipient mice (n = 8). (B) Lineage output of young or aged LT-HSCs at 4, 8, 12, 16 weeks after transplantation. Each bar represents an individual mouse. *P < 0.05. **P < 0.01.
Author response image 2
(A) Experimental design for competitive co-transplantation assay.Ten CD45.2+ young LT-HSCs and ten CD45.2+ aged LT-HSCs were transplanted with 2 × 105 CD45.1+/CD45.2+ supporting cells into lethally irradiated CD45.1+ recipient mice (n = 16). (B) Lineage output of young or aged LT-HSCs at 4, 8, 12, 16 weeks after transplantation. Each bar represents an individual mouse.
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Additional files
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Supplementary file 1
List of used antibodies.
- https://cdn.elifesciences.org/articles/95880/elife-95880-supp1-v1.xlsx
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Alteration of long- and short-term hematopoietic stem cell ratio causes myeloid-biased hematopoiesis
eLife 13:RP95880.
https://doi.org/10.7554/eLife.95880.4
